overview and status of epa collaborations for detection of

Office of Research and Development

Overview and Status of EPA collaborations for Detection of Selected Biotoxins in Drinking

Water, Soils, and Wipes

Matthew L. Magnuson, Ph.D.US Environmental Protection Agency

National Homeland Security Research Center


NHSRC Mission

To conduct research and develop scientific products that improve the capability of the Agency to carry out its homeland security responsibilities

ADVANCINGOUR NATION’S

SECURITYTHROUGHSCIENCE


Many homeland security practices may also benefit day to day operation. For example, emerging analytical techniques to monitor water quality might be used during other water emergencies and/or clean-up after contamination.

Multi Use

Homeland Security

“Normal” Environmental Operations

Cross agency

NHSRC Research Projects


NHSRC Products

• 125 reports and journal articles since 2003 (including classified)• Results presented many other ways—stakeholder meetings, symposia, workshops, etc.• Products and research plans receive rigorous quality reviews

Most scientists regarded the new streamlinedpeer-review process as ‘quite an improvement.’


Overview of Detection of Biotoxins

• Laboratory methods–SAM method compendium–Collaborative projects–Future directions


Laboratory methods

• Method development and study–Documentation: Methods and study reports

• Methods aim to have DQOs fit for their intended use by–EPA/Water Security Division through the Water Laboratory

Alliance–EPA/Office of Emergency Management through the

Environmental Response Laboratory Network (ERLN)

• Availability of Methods: Most on-going studies or in clearance–Availability announced on website and we also maintain a list

of stakeholders who are specifically informed–Register to get updates at http://www.epa.gov/nhsrc


Standardized Analytical Methods (SAM) Document

• Cornerstone document of Environmental Response Laboratory Network (ERLN)

• Identifies chemical, biological and radiological (CBR) agents of concern and preferred analytical methods to be used for ENVIRONMENTAL RESTORATIONNOT EMERGENCY RESPONSE

• Originally published in 2005, updated annually

• SAM 5.0 published in October 2009

• Searchable SAM website: www.epa.gov/sam


Generic Response Analytical Performance Considerations for Environmental Restoration

• Sample Magnitude: 10,000’s of samples for weeks to months.

• Multiple Analysis Types: Field and laboratory

• Multiple Sample Types: Clinical, Environmental, Forensic

• Multiple Sample Matrices: Clinical, food, solids, liquids, etc.


Analysis needs in Generic Response Timeline


SAM Revision 5.0 Analytical Methods

•Radiochemistry Methods•23 analytes•5 sample matrices•Qualitative determination and confirmatory methods

•Pathogen Methods•32 analytes•5 sample matrices•General remediation efficacy added using spore strips

•Biotoxin Methods•18 analytes•5 sample matrices•Presumptive, confirmatory, and biological activity methods

•Chemical Methods•135 analytes•6 sample matrices •Sample preparation and determinative methods


SAM Analytical Methods

• Sample matrix types–Solid Samples (e.g. soil)–Aqueous liquid samples (e.g. surface water, waste water, etc)–Drinking water samples–Air samples–Wipes (from surfaces)

• These are NOT compliance monitoring methods. But the goal is to meet similar DQOs as regulated analytes.


NO

YESDoes Method Meet

DQOs?

Revise Technical Approach

Write Method

Use in ERLN, WLA

Optimize Instrumentation(chromatography, mass calibrate, tune, evaluate instrument stability)

Determine Preservatives(e.g. select antimicrobial & dechlor)

Method Development Process

Goal: All steps between sample collection and data reporting

Determine Best Calibration (linear/quadratic, internal standards)

Determine Interferences (Are DQOs met in various difficult matrices?)

Determine Holding Time

Adapted from J. Shoemaker and B. Boutin, USEPA/ORD at WQTC 2008


Analytical Target Levels for Methods

–Water and Air Risk-based Exposure Guidelines Are Used to Estimate Appropriate Target Levels for Analytical Methods

–Risk-Based Criteria to Support Validation of Detection Methods for Drinking Water and Air

http://www.epa.gov/nhsrc/pubs/600r08021.pdf


• Expert input through SAM Biotoxin Working Group• Proteins

– Abrine, Ricin, Bot (A, B, E, F), Shiga 1 & 2, SEA, SEB, SEC• Small molecule

– Aflatoxin B1, α-Amanitin, anatoxin-a, brevetoxin B, α-Conotoxin, Cylindrospermopsin, Diacetoxyscirpenol, microcystins (LA, LR, LW, RR,

YR), picrotoxin, saxitoxins, T2 mycotoxin, tetrodotoxin

Detection of Biotoxins in environmental matrices—Biotoxins and SAM


• Analytical techniques– Antibody based detection schemes

• ELISA• Lateral flow devices• Various antibody-capture-release detectors (fluorescence,

electrochemiluminescence, etc.– Instrumental analysis

• Liquid chromatography-mass spectrometry• Gas chromatography-mass spectrometry• others

Detection of Biotoxins in environmental matrices –Biotoxins and SAM


Meeting throughput requirements

Multi-tier analysis approach• Environmental restoration hopefully is effective, so while there are many

samples, fewer will be positive after initial decontamination activities. • Samples initially subjected to higher throughput, screening methods• Followed by analysis of selected samples with lower throughput, but more

definitive, techniques• Application approach of techniques listed will appear in future version of

SAM (e.g. see techniques of ricin in table below)


Previous biotoxin projects

Dahlgren Naval Surface Warfare Center• Included both literature review and experimental components.• Develop an improved scientific understanding of the nature of biotoxins

potentially used to intentionally contaminate drinking water systems.• Methods of reducing the risks from biotoxin contamination• Target applications:

– Decontamination– Threat analysis


Experimental Approach

Designed to reflect possible operational paradigm during water system contamination/decontamination

• Different oxidants•chlorine, chloramine, chlorine dioxide, ozone

• Fate process•Hydrolysis, Heat inactivation, Coagulation


EPA Technology Testing and Evaluation Program (TTEP)

Test, evaluate, and report on the performance of homeland security-related technologies

• building and outdoor area decontamination• air, water, and wastewater treatment • water security technologies• detectors/monitors for air and water

Desired Outcome:

Informed decision making by our customers to detect, contain, decontaminate, and manage hazardous chemical, biological, and radiological materials purposefully or accidentally introduced into structures, facilities, drinking water systems, or the environment.

Previous biotoxin projects


Reports for various technologies atwww.epa.gov/etv

• Immunoassay Test Kits for Biotoxins– BADD Anthrax, Botulinum Toxin, and Ricin Immunoassay Test Strips – BioVerify Botulinum Toxin A and Ricin Test Kits and M-Series M1M Analyzer – EzyBot A and EzyBot B Test Kits – QTL Biosensor – RAMP Anthrax, Botulinum Toxin, and Ricin Immunoassay Test Cartridges– BioThreat Alert Anthrax, Botulinum Toxin, and Ricin Immunoassay Test Strips (Tetracore)– Anthrax, Botulinum Toxin, and Ricin Enzyme-Linked Immunosorbent Assay (ELISA,

Tetracore)

• Rapid Toxicity Testing Systems (included ricin and bot)– AbraTox Kit – Chem-IQ Tox Test Kit – IQ Toxicity Test Rapid Toxicity Testing System – ToxScreen II Rapid Toxicity Testing System (Round 1) – ToxScreen II Test Kit (Round 2) – Toxi-Chromotest– ToxTrak Rapid Toxicity Testing System – BioTox Rapid Toxicity Testing System – POLYTOX Rapid Toxicity Testing System – LuminoTox SAPS Test Kit – Eclox Rapid Toxicity Testing System


Current biotoxin detection projects

Centers for Disease Control and Prevention• The CDC’s Chemical Terrorism Analytical Response Laboratory is

currently developing assays for the quantitation of biotoxins in clinical matrices such as urine. These clinical assays are expected to becompatible with drinking water matrices.

• LC/MS/MS methods used in chemical laboratory response network (LRN) for clinical samples

• Addition of drinking water matrices to experimental design • Stability, extraction, chromatography, MS tuning• High throughput, small samples, automation, IT


Biotoxins will be extracted from water samples, separated, and analyzed

Solid Phase ExtractionHPLC

Mass SpectrometryData Analysis


Features of Adapted Methods• 100-1000 samples/day• Analytes

–α, β - amanitin–Mycotoxins–Marine Toxins –TETS–Biomarkers for ricin and abrin

• Stable Isotopic Internal Standards• Availability: Fall 2010

Native

LabeledISTD

NHHN

NH

HN

NH HN

HN

OO

O

O

O

O

O

OSO

H2NO

N

HN

OH

Exact Mass: 918.35

HOOH

HO

4,5-dihydroxyisoleucine4-hydroxyproline

asparagine

glycine

isoleucineglycine

4-hydroxytryptophan

cysteine


Current Projects

Centers for Disease Control and Prevention (very recent)• Adaptation of the LRN Botulinum Toxin ELISA assay for water. • Adaptation of the Endopep MS assay for water.

– Antibody based cleanup, SNAP-25 cleavage with MS detection• Investigate the ability of UF concentration device(s) to concentrate

botulinum toxin. • Several analytical approach adaptations for ricin.

CDCclinical matrices

EPAenvironmental

matricesSAM


Current Projects

Food Safety Inspection Service (USDA) on-going project• Adaption of immunoassay with electrochemiluminescence (ECL)

detection methods from Food Emergency Response Network (FERN) to environmental matrices

• Plant and bacterial toxins (botulinum, ricin, others)• Civil Support Teams also have this equipment and reagents


Future directions for biotoxin detection

Future collaborative work• Analysis approaches for other matrices • Additional biotoxins• Refinement of existing methods• Decontamination-focused studies (and associated detection challenges)• Enhanced federal collaborations – recent agreement between EPA,

DHS, and DOD.

Method verification• Methods may be evaluated in 2nd or more labs.• May be compatible with current public health lab operations


Acknowledgements

• Centers for Disease Control and Prevention– Rudy Johnson, Ph.D.– Jennifer Links, Ph.D.– Stephen Morse, Ph.D. – CT method

development group

• Food Safety Inspection Service– Mark Campbell, Ph.D.– Marcus Head, Ph.D.– Jim Jones, Ph.D.– Anne Hurley, DVM, MPH

• Naval Surface Warfare Center– Elaine Strauss, Ph.D.– Wynn Vo– Andrew Slaterbeck, Ph.D.– Bradford Gutting, Ph.D.

• Environmental Protection Agency– Michelle Burgess, Ph.D., OEM– Sanjiv Shah, Ph.D., NHSRC– Erin Silvestri, MPH, NHSRC– Sarah Perkins, Ph.D., NHSRC


Questions?Matthew Magnuson, Ph.D.

Research ChemistNational Homeland Security Research Center

Water Infrastructure Protection DivisionCincinnati, OH 45268

513 569 [email protected]

http://www.epa.gov/nhsrc

Disclaimer: The U.S. Environmental Protection Agency funded, partially funded, managed, and/or collaborated in the research described in this presentation. It has been subject to an administrative review but does not necessarily reflect the views of the Agency. No official endorsement should be inferred. EPA does not endorse the purchase or sale of any commercial or non-commercial products or services.

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